精品视频在线观看专区,饥渴老熟妇乱子伦视频,五月天综合婷婷综合社,国产精品美女久久久久久小说

北京伯樂(lè)生命科學(xué)發(fā)展有限公司上海辦事處 (上海天崛電子科技有限公司)

酶標(biāo)儀.超低溫冰箱.移液器.洗板機(jī).離心機(jī).二氧化碳培養(yǎng)箱.凝膠成像系統(tǒng)

食品機(jī)械設(shè)備網(wǎng)收藏該商鋪

     小標(biāo) 您所在位置:首頁(yè) > 公司動(dòng)態(tài)> 基于PCR技術(shù)的染色質(zhì)沉淀分析
產(chǎn)品搜索

請(qǐng)輸入產(chǎn)品關(guān)鍵字:

紫外分析儀/紫外可見分光光度計(jì)

熒光檢測(cè)儀

震蕩器

超聲波清洗器

儀器耗材類

試劑盒

細(xì)胞株

色譜類耗材

滅菌器

低溫冰箱

顯微鏡系統(tǒng)

PCR類

水分測(cè)定儀

恒溫干燥培養(yǎng)類

奧立龍全線產(chǎn)品

美國(guó)Bio-Rad伯樂(lè)耗材

美國(guó)Bio-rad伯樂(lè)試劑

凍干機(jī)

移液器/進(jìn)樣器

酶標(biāo)儀/洗板機(jī)

離心機(jī)

美國(guó)Bio-rad伯樂(lè)電泳系統(tǒng)

天平

純水系統(tǒng)

酸度計(jì)/離子計(jì)/電導(dǎo)計(jì)/PH計(jì)

聯(lián)系方式
地址:北京總部-〉北京市海淀區(qū)清河三街清上園4號(hào)樓B座1708
郵編:100000
聯(lián)系人:耿明
留言:在線留言
商鋪:http://m.hg0881.cn/st20882/
公司動(dòng)態(tài)

基于PCR技術(shù)的染色質(zhì)沉淀分析

點(diǎn)擊次數(shù):837 發(fā)布時(shí)間:2009-2-9

INTRODUCTION

After chromatin immunoprecipitation (ChIP), different PCR-based approaches can be used to determine how much DNA is precipitated at a locus of interest. Real-time PCR amplification is often the preferred technique. One can also use duplex PCR amplification, which is the coamplification of a fragment from the region of interesta control fragment (e.g., the actin gene, or the tubulin gene). This approach allows for estimating relative levels of specific histone modifications along chromosomal domains. For allele-specific studies (for instance, on dosage-compensation mechanisms or on genomic imprinting), electrophoretic detection of single-strand conformation polymorphisms (SSCP) or similar strategies such as hot-stop PCR can differentiate PCR products that represent the silent allele from those amplified from the active allele. If a polymorphic restriction site is present in one alleleabsent in the other, the method of choice is hot-stop PCR. If no polymorphic restriction sites are available, but there are single nucleotide polymorphisms (SNPs) that distinguish the alleles of interest, the best approach is to separate the PCR products derived from the two different alleles using SSCP. In SSCP, it is possible to discriminate denatured PCR products derived from one allele or the other because the secondary structure of each single strand will be directly dependent on the sequence itself. Hence, in nondenaturing gel conditions, each single strand will migrate differently. These four PCR-based methodologies to analyze immunoprecipitated chromatin (real-time PCR, duplex PCR, hot-stop PCR,SSCP) are presented here.

RELATED INFORMATION

Our method for chromatin immunoprecipitation (ChIP) is described in Chromatin Immunoprecipitation on Unfixed Chromatin from CellsTissues to Analyze Histone Modifications. To distinguish between alleles at loci of interest in precipitated chromatin fractions, we use "hot-stop" PCR (Steps 1-22) or SSCP (Steps 23-37). See Uejima et al. (2000) for a detailed hot-stop PCR protocol. Figure 1 provides an example of results using SSCP. To quantify how much DNA has precipitated at a locus of interest, we use real-time PCR (Steps 38-39)duplex PCR (Steps 40-53). Examples of duplex PCR analysis of precipitated chromatin are provided in Noma et al. (2001)Gregory et al. (2001).

Figure 1. Allele-specific patterns of histone modifications revealed by PCR amplificationSSCP electrophoresis. Lung tissue was dissected from a mouse that was an interspecific hybrid (H) between Mus musculus domesticus (D, paternal genome)Mus spretus (S, maternal genome). The native chromatin was then immunoprecipitated with rabbit polyclonal antisera to acetylation at lysine 9 of H3 (H3K9Ac)to dimethylation at lysine 4 of H3 (H3K4Me2; Upstate Ltd.). Radioactive PCR was performed on bound (B)unbound (U) fractions with primers that amplified from a unique sequence at an imprinting-control center located in a gene called Kvlqt1. The PCR products were denaturedsubjected to electrophoresis through a nondenaturing polyacrylamide gel (SSCP electrophoresis). The four lanes to the left show control amplifications from genomic DNAs (D, Mus musculus domesticus DNA; S, Mus spretus DNA; H, [Mus musculus domesticus x Mus spretus] F1 DNA). In the analysis of the antibody bound (B)unbound (U) fractions (right panel), the bands representing the maternalpaternal alleles are indicated.

MATERIALS

Reagents

100-bp DNA step ladder (for duplex PCR only; see Steps 40-53)

Acrylamide solution for SSCP gels (2X) (e.g., Acrylamide Solution for Mutation Detection, A5934, Sigma) (for SSCP only; see Steps 23-37)

MDE Gel Solution is a polyacrylamide-like matrix specifically optimized for SSCP.

Acrylamide/bisacrylamide (29:1 ratio; 40% stock solution) (Sigma) (for hot-stop PCR only; see Steps 1-22)

Agarose (for duplex PCR only; see Steps 40-53)

Ammonium persulfate (APS; 10%, w/v), freshly prepared (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)

α-32PdCTP (10 µCi/µl, specific activity 3000 Ci/mmol) (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)

dNTPs (stock solutions at 25 mM for each dNTP)

DNA loading buffer (6X)

30% (v/v) glycerol

0.25% (w/v) bromophenol blue

0.25% (w/v) xylene cyanol FF

Store at 4°C

Ethidium bromide solution (20 mg/ml in H2O) (for duplex PCR only; see Steps 40-53)

Forwardreverse primers (100 µM stock solutions in H2O)

For duplex PCR (Steps 40-53), primers should be designed in order to obtain comparable amplifications of the specific fragment of interestcontrol fragments (e.g., the actin gene) when using a control genomic DNA as a template. Importantly, the PCR product amplified from the region of interest should be of a size different from that amplified from the internal control region. This allows the two different PCR products to be distinguished by agarose gel electrophoresis.

PCR amplification buffer (10X) (supplied with the Taq DNA polymerase)

Reagents for Real-Time PCR (for real-time PCR only; see Steps 38-39)

Restriction endonuclease (for hot-stop PCR only; see Steps 1-22)

This endonuclease must be specific for a polymorphic restriction site within the amplified DNA fragment.

Restriction endonuclease buffer (10X) (supplied with the restriction endonuclease; for hot-stop PCR only; see Steps 1-22)

SSCP loading dye (for SSCP only; see Steps 23-37)

95% (v/v) formamide

10 mM NaOH

0.25% (w/v) bromophenol blue

0.25% (w/v) xylene cyanol

Taq DNA polymerase (5 U/µl)

TBE Buffer (1X5X)

Prepare a 5x stock solution in 1 liter of H2O:

54 g of Tris base

27.5 g of boric acid

20 ml of 0.5 M EDTA (pH 8.0)

The 0.5x working solution is 45 mM Tris-borate/1 mM EDTA.

TBE is usually madestored as a 5x or 10x stock solution. The pH of the concentrated stock buffer should be approx. 8.3. Dilute the concentrated stock buffer just before usemake the gel solutionthe electrophoresis buffer from the same concentrated stock solution. Some investigators prefer to use more concentrated stock solutions of TBE (10x as opposed to 5x). However, 5x stock solution is more stable because the solutes do not precipitate during storage. Passing the 5x or 10x buffer stocks through a 0.22-µm filter can prevent or delay formation of precipitates.

Template DNA This is the genomic DNA extracted from the antibody-boundantibody-unbound fractions (from Chromatin Immunoprecipitation on Unfixed Chromatin from CellsTissues to Analyze Histone Modifications). Control genomic DNA should be used as well. For each PCR reaction, we use 20-50 ng of template DNA.

N,N,N'',N''-Tetramethyl-ethylenediamine (TEMED) (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)

[ 打印 ] [ 返回頂部 ] [ 關(guān)閉

| 商鋪首頁(yè) | 公司檔案 | 產(chǎn)品展示 |公司動(dòng)態(tài) | 詢價(jià)留言 | 聯(lián)系我們 | 會(huì)員管理 |
食品機(jī)械設(shè)備網(wǎng) 設(shè)計(jì)制作,未經(jīng)允許翻錄必究.Copyright(C) http://m.hg0881.cn, All rights reserved.
以上信息由企業(yè)自行提供,信息內(nèi)容的真實(shí)性、準(zhǔn)確性和合法性由相關(guān)企業(yè)負(fù)責(zé),食品機(jī)械設(shè)備網(wǎng)對(duì)此不承擔(dān)任何保證責(zé)任。
溫馨提示:為規(guī)避購(gòu)買風(fēng)險(xiǎn),建議您在購(gòu)買產(chǎn)品前務(wù)必確認(rèn)供應(yīng)商資質(zhì)及產(chǎn)品質(zhì)量。
二維碼

掃一掃訪問(wèn)手機(jī)站